Journal oflmmunological Methods, 13 (1976) 321--331 © Elsevier/North-Holland Biomedical Press
321
PARTIAL PURIFICATION OF HUMAN LEUCOCYTE MIGRATION I N H I B I T O R Y F A C T O R ( L I F ) BY I M M U N O S O R P T I O N O F S U P E R N A T A N T P R O T E I N C O N T A M I N A N T S D E T E C T E D BY C R O S S E D IMMUNOELECTROPHORESIS
KLAUS BENDTZEN
The Laboratory of Clinical Immunology, Medical Department TA, Rigshospitalet University Hospital, Tagensvej 18, DK-2200 Copenhagen N, Denmark (Received 29 March 1976, accepted 20 July 1976)
Concentrated supernatants from washed human lymphocytes incubated in serum-free medium were investigated by crossed immunoelectrophoresis. Quantitatively, the most important macromolecules were serum proteins, in particular albumin and degraded products of albumin. No gross difference was detectable between supernatants from concanavalin A stimulated and unstimulated lymphocytes. The degraded proteins were 6onsidered to arise as a result of proteolytic enzymes present in both stimulated and unstimulated lymphocyte supernatants. These molecules exhibited almost the same electrophoretic mobility and molecular weight as native albumin, and might therefore be expected to be difficult to separate from some lymphokines by conventional biochemical techniques. Rabbit immunoglobulins to whole human serum proteins together with immunoglobulins against crude supernatants of mitogen stimulated lymphocytes were therefore bound covalently to an agarose matrix. This preparation efficiently removed all detectable proteins from concentrated supernatants of activated lymphocytes as determined by crossed immunoelectrophoresis. Leucocyte migration inhibitory factor (LIF) was recovered almost quantitatively, and a 40-fold purification of LIF was achieved. The technique is rapid, economical and well suited as an initial step for purification of large quantities of LIF. INTRODUCTION T- a n d B - l y m p h o c y t e s m e d i a t e c e l l u l a r i m m u n i t y e i t h e r d i r e c t l y or b y secreting biologically active p r o d u c t s : L y m p h o k i n e s . I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f l y m p h o k i n e s has b e e n e x t r e m e l y difficult because of the small a m o u n t s p r o d u c e d both by antigen- and mitogen stimulated l y m p h o c y t e s . Thus, c o n t a m i n a t i o n by a variety of other proteins even in v e r y l i m i t e d q u a n t i t i e s s e r i o u s l y h a m p e r s t h e e f f o r t s t o p u r i f y l y m p h o k i n e s b y c o n v e n t i o n a l b i o c h e m i c a l t e c h n i q u e s . F u r t h e r m o r e w h e n emp l o y i n g a c o m b i n a t i o n o f p r o c e d u r e s s u c h as gel f i l t r a t i o n , i o n - e x c h a n g e c h r o m a t o g r a p h y , f r a c t i o n a l p r e c i p i t a t i o n w i t h a m m o n i u m s u l p h a t e a n d disc e l e c t r o p h o r e s i s ( R e m o l d e t al., 1 9 7 0 ; D u m o n d e e t al., 1 9 7 2 ; G r a n g e r e t al., 1 9 7 3 ) t h e y i e l d o f a c t i v e m a t e r i a l is v e r y low. To proceed from the partial purification already achieved to the eventual
322 isolation and characterization, in chemical terms, of lymphokines such as human leucocyte migration inhibitory factor (LIF) (Rocklin, 1975), other techniques appear to be necessary, and our knowledge of the macromolecules contaminating lymphokine preparations must be further extended. The aim of this paper is to present the results of an immunochemical study of highly concentrated LIF-rich material by means of crossed immunoelectrophoresis (CIE). These results have been used to develop a simple procedure based on affinity chromatography for the immunological removal of contaminants of human LIF preparations. MATERIALS AND METHODS
Lymphocyte cultures Peripheral blood mononuclear cells were obtained from normal adults as previously described (Bendtzen et al., 1975). The cells were washed three times with Hank's balanced salt solution, and suspensions of 2.5 X 106 cells/ ml were made in serum-free medium TC-199 (Difco Labor., Michigan, U.S.A.). Cell suspensions were incubated in the presence (active supernatant) or in the absence (control supernatant) of concanavalin A 80 pg/ml (Pharmacia, Uppsala, Sweden) (Con A). After 22 h at 37°C in a 2% CO2: 98% air atmosphere the supernatants were harvested, and the control supernatant was reconstituted with Con A 80 pg/ml. Crude supernatants were desalted and depleted of Con A by passage through small columns of Sephadex G-100 (Parmacia) (Bendtzen et al., 1975) equilibrated in volatile buffer (0.05 M ammonium bicarbonate; acetic acid, pH 7.2, 0.1 mM CaC12, 0.1 mM MgC12, 0.1 mM MnC12). The columns were calibrated to retain only molecules with molecular weights less than 10,000 daltons. The column eluates were passed through washed Millipore filters (0.45 pm pore size), lyophilized and stored a t - - 2 0 ° C (crude supernatants).
Sephadex G-I O0 fractionation of lymphocyte supernatants Fractionation on large Sephadex G-100 columns was carried out as previously detailed (Bendtzen, 1975). In brief, lyophilized crude supernatants were pooled in 1 : 70th the original volume and chromatographed on Sephadex G-100 glass columns (1.15 X 90 mm) in volatile buffer. Fractions containing molecules of molecular weight between 40,000 and 80,000 daltons were pooled, lyophilized and stored at 4 ° C.
Assay for LIF The indirect leucocyte migration agarose technique originally described by Clausen (1972) and modified by Bendtzen et al. (1975) was employed using unrelated peripheral blood leukocytes as migratory cells. 22 X 106 cells/90 pl
323 culture supernatant were tested in 7 #l aliquots for migration under agarose and a migration index (MI) was determined: MI =
Mean area of migration in the presence of LIF-supernatant Mean area of migration in the presence of control supernatant
Mean area was determined by quadruplicate tests. A n tibody preparations
Purified antibodies against LIF-rich crude supernatants (anti-sup), i.e. the immunoglobulin fraction of the antiserum, were prepared by immunization of two rabbits over a period of four months following the immunization scheme proposed by Harboe and Ingild (1973). Each rabbit received a total of approximately 1.5 mg protein present in 60 ml whole supernatant from Con A stimulated lymphocytes. The mitogen-free lyophilized material was concentrated fifty times and emulsified with an equal volume of incomplete Freunds adjuvant immediately prior to injection in the animals. Rabbit antibodies against human serum proteins (code 100 SF) were obtained from Dakopatts A/S, Copenhagen, Denmark, who also supplied the other monospecific absorbed antibodies against human serum proteins. Antibodies were preserved in 15 mM NaN3 solution and stored at 4 ° C. In some experiments 50 U/ml of aprotinin (Novo Industrie GmbH, Mainz, West Germany) was added to the antibody preparations. Im m unoe lec trop horesis
Crossed immunoelectrophoresis (CIE) (Weeke, 1973b) and CIE with intermediate gel (Axelsen, 1973) were carried out in 1% agarose gels (Litex, Glostrup, Denmark) in a buffer of 0.073 M Tris, 0.0245 M barbital, 0.0003 M calcium lactate and 0.003 M sodium azide (pH 8.6). The first dimension electrophoresis was run at 14°C applying 10 V/cm for 40 min (5 X 5 and 5 X 7 cm plates) and for 80 min (10 X 10 cm plates). The second dimension electrophoresis was performed at 2 V/cm for 22 h in gels containing antibodies as detailed in the figures. Drying, washing and staining of the plates was carried out as described by Weeke (1973a). In some experiments the non-ionic detergent Berol EMU-043 (Modokemi AB, Stennungsund, Sweden) was added to the gel in a concentration of 1% w/v. All experiments were repeated on at least four different l y m p h o c y t e culture supernatants. Preparation o f affinity columns
All procedures were carried out at 22°C unless otherwise indicated. Four g activated CH-Sepharose 4B (Pharmacia) was swollen in 0.001 M HC1 and washed in a glass column with 60 ml of the same solution followed by 20 ml 0.1 M NaHCO3, pH 8.0.
324 5 ml anti-sup, m i x e d with 5 ml o f purified r a b b i t a n t i b o d i e s t o h u m a n serum p r o t e i n s (Code 1 0 0 SF Dakopmtts A/S, C o p e n h a g e n , D e n m a r k ) was adjusted t o pH 8.0 with 0.1 M N a O H and the m i x t u r e was r u n into the gel. C o u p l i n g o f the i m m u n o g l o b u l i n s to the gel was allowed to take place at 3 7 ° C for 1 h with gentle agitation o f the c o l u m n . R e m a i n i n g active g r o u p s were b l o c k e d b y i n c u b a t i n g the p r o d u c t in 0.05 M Tris buffer, pH 8.0, 0.5 M NaC1 (15 ml) for 1 h. The gel was t h e n w a s h e d with 0.05 M Tris buffer, pH 8.0 (20 ml), 0.05 M a c e t a t e buffer, pH 4.0 (20 ml) and 0.1 M p h o s p h a t e b u f f e r , p H 7.2 (20 ml), each c o n t a i n i n g 0.5 M NaC1. Finally, 6 ml gel suspension was p o u r e d into each o f t w o c o l u m n s (K 9 / 1 5 - P h a r m a c i a ) and stored at 4°C until use. T h e c o l u m n s were r e g e n e r a t e d a f t e r use b y washing w i t h 0 . 0 0 2 M glycine/ HC1 b u f f e r , pH 2.8 (20 ml), 0.05 M Tris buffer, p H 8.0 (12 ml) and 0.1 M p h o s p h a t e buffer, pH 7.2 (12 ml), each c o n t a i n i n g 0.5 M NaC1.
TABLE 1 LIF activities and total protein contents of supernatants before and after affinity chromatography for removal of lymphokine contaminants. Exp. no.
1
Original Passage super- through nate columns volume (ml) 200 (200)
Before After
2
120 (120)
Before After
3
145 (145)
Before After
4
145 (145)
Before After
Mean 1--4
Before After
Protein content (rag)
Ratio
Migration index Supernatant concentration when assayed for LIF x8 x4 x2
6.51 (5.90) 0.28 (0.22)
23 : 1 (27 : 1)
5.80 (4.60) 0.11 (0.08)
53 : 1 (57:1)
4.00 (3.00) 0.10 (0.08)
40 : 1 (37:1)
5.20 (4.98) 0.17 (0.12)
31 : 1 (41:1)
0.70
0.77
0.79
0.75
0.75
0.83
0.53
0.75
0.87
0.55
0.75
0.90
0.54
0.70
0.92
0.56
0.72
0.94
0.56
0.73
0.78
0.61
0.73
0.83
0.58 ± 0.08 0.62 ± 0.09 (ns) *
0.74 ÷ 0.03 0.74 ± 0.02 (ns)
* Values in parenthesis represent the control supernatants. P > 0.05 was considered not significant .(Wilcoxon rank sum test).
0.84 ± 0.07 0.87 ± 0.05 (ns)
325 I m m u n o s o r p tion o f con tamina ting pro teins Lyophilized, pooled Sephadex G-100 fractions containing LIF were dissolved in 2 ml 0.1 M phosphate buffer, pH 7.2, 0.5 M NaC1, applied to the immunoglobulin-Sepharose column and eluted with the same buffer at a flow rate of 0.5 ml/min. An identical column was run in parallel with pooled fractions from Sephadex G-100 chromatographed control supernatant. The initial 2.5 ml eluate was discarded and the following 8 ml were collected, desalted on Sephadex G-50 and lyophilized. Lyophilized material from both chromatographed and non-chromatographed supernatants was reconstituted in medium TC-199 (Difco Labor., Michigan, U.S.A.) containing 10% horse serum to give the concentrations of the original supernatants outlined in table 1. These preparations were tested for LIF activity on the same indicator cell population. Protein measurements The method of L o w r y et al. (1951) was employed using human serum albumin as standard. RESULTS Immunoelectrophoresis Fig. l a shows the immunoprecipitation pattern obtained when concentrated lymphokine containing crude supernatant was run in CIE against antisup. Essentially the same pattern was seen when control supernatants were tested. The two sharp and normally stained precipitates migrate together with and anodic of albumin in the first dimension and show the characteristic features of partial immunochemical identity. The unending anodic arc of the blurred precipitate (b) is also indicative of partial identity with c. Three precipitates with a and ~ mobility are distinguishable b u t stain poorly and diffusely. Precipitates with double peaks, unending and blurred precipitates and changes in migration velocity are all features of proteolytic degradation (Bjerrum and B~bg-Hansen, 1975). Since the use of aprotinin, a protease inhibitor, has been shown to inhibit an artefact in CIE caused by proteolytic activity in antibody preparations (Bjerrum et al., 1975) several experiments were carried o u t with and w i t h o u t aprotinin in the antibody solution. This, however, did n o t change the precipitation pattern. When performing CIE with gels containing the non-ionic detergent Berol, the findings were also unchanged. The visible effects of proteolytic degradation did n o t increase as a function of time, since identical precipitation patterns were obtained when testing supernatants after 6 h, 24 h and 72 h at 23°C following the initial culture period.
326
When concentrated supernatants from both stimulated and unstimulated l y m p h o c y t e s were run against an antibody preparation against human serum proteins eleven precipitates appeared (fig. l b ) . Using CIE with intermediate gels containing monospecific antibodies against serum proteins all except three precipitates were identified. It appeared that only albumin was degraded by proteolytic activity (fig.2). These degraded molecules shared at least two antigenic determinants recognized by the monospecific antibody against native albumin. This antibody preparation also contained immunoglobulins against the three antigens visible in a, b and c in fig. la. When subjected to Sephadex G-100 column chromatography all three components eluted in the same fractions corresponding to a molecular weight of 70,000 daltons. The anti-sup, preparation did not contain detectable antibodies to super-
q
.B
Fig. 1. Crossed immunoelectrophoresis of concentrated serum-free supernatants from human lymphocytes stimulated with Con A. A. Antigen: 20 pl supernatant (x 50). Antibodies: 15 pl/cm 2 of rabbit antibodies against human lymphocyte supernatant. B. Antigen: 15 pl supernatant.(× 100). Antibodies: 7.5 pl/cm 2 of pooled rabbit antibodies against human serum proteins. The following precipitates were identified: prealbumin (p), albumin (a, b and c), orosomucoid (o), alfal-antitrypsin (at), haptoglobin (h) and transferrin (t). The arrows indicate the features of partial immunochemical identity. The 5 × 5 cm plates were stained with Coomassie Brilliant Blue R.
327
Fig. 2. Crossed immunoelectrophoresis of 15 pl concentrated (X 100) serum-free supernatants from human lymphocytes stimulated with Con A. A. Blank intermediate gel. B. Intermediate gel containing absorbed monospecific antibodies against human albumin (5 pl/cm2). The reference gels contained antibodies against human serum proteins (7.5 pl/cm2). Precipitates of albumin and degraded products of albumin are marked a, b and c. The 5 X 7 cm plates were stained with Coomassie Brilliant Blue R.
n a t a n t p r o t e i n s which were n o t p r e s e n t in c o m m e r c i a l anti-whole serum protein (fig. 3). H o w e v e r , separate e x p e r i m e n t s s h o w e d t h a t the a n t i b o d y titer o f anti-sup, against c o m p o n e n t c (fig. l a ) s e e m e d to be m u c h higher t h a n t h a t o f anti-whole serum p r o t e i n . T h e p r o t e i n c o n c e n t r a t i o n o f u n c o n c e n t r a t e d s u p e r n a t a n t s f r o m b o t h stim u l a t e d and u n s t i m u l a t e d l y m p h o c y t e s was 38 p g / m l (standard deviation: 9 ttg/ml, 6 e x p e r i m e n t s ) and 33 p g / m l (standard deviation: 7 pg/ml, 6 experim e n t s ) respectively. T h u s , the c o n c e n t r a t i o n o f i d e n t i f i e d p r o t e i n s o t h e r t h a n native and degraded albumin m u s t be v e r y low. H o w e v e r , a n t i b o d i e s against some o f these m o l e c u l e s were p r o d u c e d b y the i m m u n i z e d animals (fig. 4).
Immunosorption of protein contaminants F o u r d i f f e r e n t e x p e r i m e n t s were carried o u t using p o o l e d Con A stimulated s u p e r n a t a n t s c o r r e s p o n d i n g t o original v o l u m e s o f 145 ml to 200 ml. E q u a l a m o u n t s o f c o n t r o l s u p e r n a t a n t s were r u n in parallel.
328
Fig. 3. Crossed i m m u n o e l e c t r o p h o r e s i s of 20 ~l c o n c e n t r a t e d (× 50) serum-free supernatants f r o m h u m a n l y m p h o c y t e s stimulated with Con A. The intermediate gel contained antibodies against h u m a n serum proteins (10 pl/cm2), and the reference gel contained antibodies against h u m a n l y m p h o c y t e supernatant (15 pl/cm2). No precipitates are detectable in the reference gel. The 5 × 7 cm plate was stained with Coomassie Brilliant Blue R.
A
B
Fig. 4. Crossed i m m u n o e l e c t r o p h o r e s i s of h u m a n serum. A. Blank intermediate gel. B. I n t e r m e d i a t e gel containing antibodies against h u m a n l y m p h o c y t e supernatant (50 pl/cm2). The reference gels contained antibodies against h u m a n serum proteins (17 /al/ cm2). It is shown, that the intermediate gel in B contain antibodies against the following serum proteins (arrows): albumin (a), o r o s o m u c o i d (o), a l f a r a n t i t r y p s i n (at), haptoglobin (h) and transferrin (t). The 10 × 10 cm plates were stained with Coomassie Brilliant Blue R.
329
4:,
Fig. 5. Crossed immunoelectrophoresis of pooled, LIF-rich, Sephadex G-100 fractions before (A) and after (B) affinity chromatography for immunological removal of contaminating proteins. A. Antigen: 152pl pooled Sephadex G-100 fractions (× 70 original supernatant). Antibodies: 7.5 pl/cm of pooled rabbit antibodies against human serum proteins. B. Antigen: 15 pl affinity chromatographed solution (X 70 original supernatant). Antibodies: As in 1A. The 5 X 5 cm plates were stained with Coomassie Brilliant Blue R.
The recovery of LIF activity could be estimated by testing stepwise concentrated chromatographed and non-chromatographed material on the same indicator cell population. As shown in table 1 a high yield of LIF was obtained after immunosorption of contaminating proteins. LIF activity could n o t be demonstrated in control supernatants when compared with cells migrating in medium alone. The protein contents of both active and control supernatants are also shown in table 1. Following immunosorption of contaminants the total amount of protein in the active supernatants was reduced by a factor of 23 to 53. Approximately the same relative reduction in protein contents of active and control supernatants was found. Crossed immunoelectrophoresis of Sephadex G-100 chromatographed, LIF-rich material run against a balanced antibody preparation to human serum proteins revealed two major reactions and one very faint precipitate (fig. 5a). These precipitates were identified as albumin and degraded products of albumin. Passage through the immunoglobulin-Sepharose column removed all three precipitating proteins (fig. 5b). DISCUSSION
The partial purification of human LIF seems to present even more difficulties than encountered during the attempts to purify guinea pig MIF (Remold et al., 1970; Rocklin, 1975). This is mainly because LIF, unlike MIF, cannot be separated from albumin because of their similarity in molecular weight and electrophoretic mobility. Proteolytic enzymes are frequently active during the preparation of tissue proteins (Barrett and Dingle, 1971). Supernatants of antigen-stimulated lym-
330
phocytes also contain proteases, and an esterase associated with MIF and LIF activity has been proposed (Haveman et al., 1972; Remold, 1974). Since some lymphokines are unstable in solution even when frozen, degradation by contaminating proteases is possible, and measures to inhibit these enzymes might be beneficial in preserving lymphokines. However, the expression of human LIF activity seems to be inhibited b y some animal and plant antiproteases and phenylmethyl sulfonyl fluoride (Bendtzen, 1976). The use of antiproteases in l y m p h o c y t e cultures is therefore n o t feasible for obtaining more stable lymphokine preparations. The experiments reported here were initiated in order to provide more information on the nature of contaminants with special emphasis towards proteins with similar electrophoretic mobility to that of LIF. With this information an affinity chromatography technique was developed for rapid removal of all detectable protein contaminants. Since even very small amounts of supernatant proteins gave rise to antib o d y production undetected proteins must contribute little to the total amount of contaminants. N o t all proteins, however, may induced the formation of precipitating antibodies and be demonstrable by immunoelectrophoresis, but the use of affinity column chromatography might nevertheless remove all contaminants to which an antibody response occured. Since antibodies to LIF were apparently n o t elicited by the immunization schedule used, such a technique could prove useful in attempts to purify LIF. Immunoglobulins to whole serum proteins and antibodies against crude LIFrich supernatants were therefore covalently b o u n d to a beaded agarose matrix. The latter immunoglobulins were employed because they contained higher titers against albumin c o m p o n e n t c (fig. 1). As shown, the technique provides an estimated 40-fold purification of LIF, b u t even more important is the very high yield of active material making this procedure well suited as an initial step in LIF purification. The c~lumn capacity for removing contaminants is sufficiently high to enable very small columns to be used. This presumably contributes to the high recovery of mediator activity by reducing the amount of unspecifically adsorbed material. The procedure is rapid and the risk of protein denaturation seems negligible. Furthermore, the experiments can be carried out at low cost since the columns can be regenerated and used several times. Thus, the principle of direct immunological removal of contaminating protein seems promising for obtaining high purity lymphokine preparations. It must however be realized that the technique presented here has limited efficiency, and should be combined with other methods in attempts to isolate specific lymphokines. ACKNOWLEDGEMENTS The study was supported by grants from the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Greenland and the Faroe
331
Islands, the Danish Medical Research Council and the Danish Foundation for the Advancement of Medical Science. I thank the staff of The Protein Laboratory, University of Copenhagen for valuable aid and Mrs. Lidy Broersma for expert technical assistance. REFERENCES Axelsen, N.H., 1973, Scand. J. Immunol. 2, Suppl. 1, 71. Barrett, A.J. and J. Dingle, 1971, Tissue Proteinases (North-Holland, Amsterdam-London). Bendtzen, K., 1975, Acta Allergol. 30, 327. Bendtzen, K., 1976, Acta Pathol. Microbiol. Scand. Sect. C. (in press). Bendtzen, K., V. Andersen and G. Bendixen, 1975, Acta Allergol. 30, 133. Bjerrum, O.J. and T.C. BOg-Hansen, 1975, Scand. J. Immunol. 4, Suppl. 2, 89. Bjerrum, O.J., J. Ramlau, I. Clemmesen, A. Ingild and T.C. B~bg-Hansen, 1975, Scand. J. Immunol. 4, Suppl. 2, 81. Clausen, J.E., 1972, J. Immunol. 108,453. Dumonde, D.C., D.A. Page, M. Matthew and R.A. Wolstencroft, 1972, Clin. Exp. Immunol. 10, 25. Granger, G.A., E.C. Laserna, W.P. Kolb and F. Chapman, 1973, Proc. Natl. Acad. Sci. 70, 27. Harboe, N. and A. Ingild, 1973, Scand. J. Immunol. 2, Suppl. 1, 161. Havemann, K., M. Horvat, C.-P. Sodomann, K. Havemann and S. Biirger, 1972, Eur. J. Immunol. 2, 97. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Remold, H.G., 1974, J. Immunol. 112, 1571. Remold, H.G., A.B. Katz, E. Haber and J.R. David, 1970, Cell. Immunol. 1,133. Rocklin, R.E., 1975, J. Immunol. 114, 1161. Weeke, B., 1973a, Scand. J. Immunol. 2, Suppl. 1, 15. Weeke, B., 1973b, Scand. J. Immunol. 2, Suppl. 1, 47.
321
PARTIAL PURIFICATION OF HUMAN LEUCOCYTE MIGRATION I N H I B I T O R Y F A C T O R ( L I F ) BY I M M U N O S O R P T I O N O F S U P E R N A T A N T P R O T E I N C O N T A M I N A N T S D E T E C T E D BY C R O S S E D IMMUNOELECTROPHORESIS
KLAUS BENDTZEN
The Laboratory of Clinical Immunology, Medical Department TA, Rigshospitalet University Hospital, Tagensvej 18, DK-2200 Copenhagen N, Denmark (Received 29 March 1976, accepted 20 July 1976)
Concentrated supernatants from washed human lymphocytes incubated in serum-free medium were investigated by crossed immunoelectrophoresis. Quantitatively, the most important macromolecules were serum proteins, in particular albumin and degraded products of albumin. No gross difference was detectable between supernatants from concanavalin A stimulated and unstimulated lymphocytes. The degraded proteins were 6onsidered to arise as a result of proteolytic enzymes present in both stimulated and unstimulated lymphocyte supernatants. These molecules exhibited almost the same electrophoretic mobility and molecular weight as native albumin, and might therefore be expected to be difficult to separate from some lymphokines by conventional biochemical techniques. Rabbit immunoglobulins to whole human serum proteins together with immunoglobulins against crude supernatants of mitogen stimulated lymphocytes were therefore bound covalently to an agarose matrix. This preparation efficiently removed all detectable proteins from concentrated supernatants of activated lymphocytes as determined by crossed immunoelectrophoresis. Leucocyte migration inhibitory factor (LIF) was recovered almost quantitatively, and a 40-fold purification of LIF was achieved. The technique is rapid, economical and well suited as an initial step for purification of large quantities of LIF. INTRODUCTION T- a n d B - l y m p h o c y t e s m e d i a t e c e l l u l a r i m m u n i t y e i t h e r d i r e c t l y or b y secreting biologically active p r o d u c t s : L y m p h o k i n e s . I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f l y m p h o k i n e s has b e e n e x t r e m e l y difficult because of the small a m o u n t s p r o d u c e d both by antigen- and mitogen stimulated l y m p h o c y t e s . Thus, c o n t a m i n a t i o n by a variety of other proteins even in v e r y l i m i t e d q u a n t i t i e s s e r i o u s l y h a m p e r s t h e e f f o r t s t o p u r i f y l y m p h o k i n e s b y c o n v e n t i o n a l b i o c h e m i c a l t e c h n i q u e s . F u r t h e r m o r e w h e n emp l o y i n g a c o m b i n a t i o n o f p r o c e d u r e s s u c h as gel f i l t r a t i o n , i o n - e x c h a n g e c h r o m a t o g r a p h y , f r a c t i o n a l p r e c i p i t a t i o n w i t h a m m o n i u m s u l p h a t e a n d disc e l e c t r o p h o r e s i s ( R e m o l d e t al., 1 9 7 0 ; D u m o n d e e t al., 1 9 7 2 ; G r a n g e r e t al., 1 9 7 3 ) t h e y i e l d o f a c t i v e m a t e r i a l is v e r y low. To proceed from the partial purification already achieved to the eventual
322 isolation and characterization, in chemical terms, of lymphokines such as human leucocyte migration inhibitory factor (LIF) (Rocklin, 1975), other techniques appear to be necessary, and our knowledge of the macromolecules contaminating lymphokine preparations must be further extended. The aim of this paper is to present the results of an immunochemical study of highly concentrated LIF-rich material by means of crossed immunoelectrophoresis (CIE). These results have been used to develop a simple procedure based on affinity chromatography for the immunological removal of contaminants of human LIF preparations. MATERIALS AND METHODS
Lymphocyte cultures Peripheral blood mononuclear cells were obtained from normal adults as previously described (Bendtzen et al., 1975). The cells were washed three times with Hank's balanced salt solution, and suspensions of 2.5 X 106 cells/ ml were made in serum-free medium TC-199 (Difco Labor., Michigan, U.S.A.). Cell suspensions were incubated in the presence (active supernatant) or in the absence (control supernatant) of concanavalin A 80 pg/ml (Pharmacia, Uppsala, Sweden) (Con A). After 22 h at 37°C in a 2% CO2: 98% air atmosphere the supernatants were harvested, and the control supernatant was reconstituted with Con A 80 pg/ml. Crude supernatants were desalted and depleted of Con A by passage through small columns of Sephadex G-100 (Parmacia) (Bendtzen et al., 1975) equilibrated in volatile buffer (0.05 M ammonium bicarbonate; acetic acid, pH 7.2, 0.1 mM CaC12, 0.1 mM MgC12, 0.1 mM MnC12). The columns were calibrated to retain only molecules with molecular weights less than 10,000 daltons. The column eluates were passed through washed Millipore filters (0.45 pm pore size), lyophilized and stored a t - - 2 0 ° C (crude supernatants).
Sephadex G-I O0 fractionation of lymphocyte supernatants Fractionation on large Sephadex G-100 columns was carried out as previously detailed (Bendtzen, 1975). In brief, lyophilized crude supernatants were pooled in 1 : 70th the original volume and chromatographed on Sephadex G-100 glass columns (1.15 X 90 mm) in volatile buffer. Fractions containing molecules of molecular weight between 40,000 and 80,000 daltons were pooled, lyophilized and stored at 4 ° C.
Assay for LIF The indirect leucocyte migration agarose technique originally described by Clausen (1972) and modified by Bendtzen et al. (1975) was employed using unrelated peripheral blood leukocytes as migratory cells. 22 X 106 cells/90 pl
323 culture supernatant were tested in 7 #l aliquots for migration under agarose and a migration index (MI) was determined: MI =
Mean area of migration in the presence of LIF-supernatant Mean area of migration in the presence of control supernatant
Mean area was determined by quadruplicate tests. A n tibody preparations
Purified antibodies against LIF-rich crude supernatants (anti-sup), i.e. the immunoglobulin fraction of the antiserum, were prepared by immunization of two rabbits over a period of four months following the immunization scheme proposed by Harboe and Ingild (1973). Each rabbit received a total of approximately 1.5 mg protein present in 60 ml whole supernatant from Con A stimulated lymphocytes. The mitogen-free lyophilized material was concentrated fifty times and emulsified with an equal volume of incomplete Freunds adjuvant immediately prior to injection in the animals. Rabbit antibodies against human serum proteins (code 100 SF) were obtained from Dakopatts A/S, Copenhagen, Denmark, who also supplied the other monospecific absorbed antibodies against human serum proteins. Antibodies were preserved in 15 mM NaN3 solution and stored at 4 ° C. In some experiments 50 U/ml of aprotinin (Novo Industrie GmbH, Mainz, West Germany) was added to the antibody preparations. Im m unoe lec trop horesis
Crossed immunoelectrophoresis (CIE) (Weeke, 1973b) and CIE with intermediate gel (Axelsen, 1973) were carried out in 1% agarose gels (Litex, Glostrup, Denmark) in a buffer of 0.073 M Tris, 0.0245 M barbital, 0.0003 M calcium lactate and 0.003 M sodium azide (pH 8.6). The first dimension electrophoresis was run at 14°C applying 10 V/cm for 40 min (5 X 5 and 5 X 7 cm plates) and for 80 min (10 X 10 cm plates). The second dimension electrophoresis was performed at 2 V/cm for 22 h in gels containing antibodies as detailed in the figures. Drying, washing and staining of the plates was carried out as described by Weeke (1973a). In some experiments the non-ionic detergent Berol EMU-043 (Modokemi AB, Stennungsund, Sweden) was added to the gel in a concentration of 1% w/v. All experiments were repeated on at least four different l y m p h o c y t e culture supernatants. Preparation o f affinity columns
All procedures were carried out at 22°C unless otherwise indicated. Four g activated CH-Sepharose 4B (Pharmacia) was swollen in 0.001 M HC1 and washed in a glass column with 60 ml of the same solution followed by 20 ml 0.1 M NaHCO3, pH 8.0.
324 5 ml anti-sup, m i x e d with 5 ml o f purified r a b b i t a n t i b o d i e s t o h u m a n serum p r o t e i n s (Code 1 0 0 SF Dakopmtts A/S, C o p e n h a g e n , D e n m a r k ) was adjusted t o pH 8.0 with 0.1 M N a O H and the m i x t u r e was r u n into the gel. C o u p l i n g o f the i m m u n o g l o b u l i n s to the gel was allowed to take place at 3 7 ° C for 1 h with gentle agitation o f the c o l u m n . R e m a i n i n g active g r o u p s were b l o c k e d b y i n c u b a t i n g the p r o d u c t in 0.05 M Tris buffer, pH 8.0, 0.5 M NaC1 (15 ml) for 1 h. The gel was t h e n w a s h e d with 0.05 M Tris buffer, pH 8.0 (20 ml), 0.05 M a c e t a t e buffer, pH 4.0 (20 ml) and 0.1 M p h o s p h a t e b u f f e r , p H 7.2 (20 ml), each c o n t a i n i n g 0.5 M NaC1. Finally, 6 ml gel suspension was p o u r e d into each o f t w o c o l u m n s (K 9 / 1 5 - P h a r m a c i a ) and stored at 4°C until use. T h e c o l u m n s were r e g e n e r a t e d a f t e r use b y washing w i t h 0 . 0 0 2 M glycine/ HC1 b u f f e r , pH 2.8 (20 ml), 0.05 M Tris buffer, p H 8.0 (12 ml) and 0.1 M p h o s p h a t e buffer, pH 7.2 (12 ml), each c o n t a i n i n g 0.5 M NaC1.
TABLE 1 LIF activities and total protein contents of supernatants before and after affinity chromatography for removal of lymphokine contaminants. Exp. no.
1
Original Passage super- through nate columns volume (ml) 200 (200)
Before After
2
120 (120)
Before After
3
145 (145)
Before After
4
145 (145)
Before After
Mean 1--4
Before After
Protein content (rag)
Ratio
Migration index Supernatant concentration when assayed for LIF x8 x4 x2
6.51 (5.90) 0.28 (0.22)
23 : 1 (27 : 1)
5.80 (4.60) 0.11 (0.08)
53 : 1 (57:1)
4.00 (3.00) 0.10 (0.08)
40 : 1 (37:1)
5.20 (4.98) 0.17 (0.12)
31 : 1 (41:1)
0.70
0.77
0.79
0.75
0.75
0.83
0.53
0.75
0.87
0.55
0.75
0.90
0.54
0.70
0.92
0.56
0.72
0.94
0.56
0.73
0.78
0.61
0.73
0.83
0.58 ± 0.08 0.62 ± 0.09 (ns) *
0.74 ÷ 0.03 0.74 ± 0.02 (ns)
* Values in parenthesis represent the control supernatants. P > 0.05 was considered not significant .(Wilcoxon rank sum test).
0.84 ± 0.07 0.87 ± 0.05 (ns)
325 I m m u n o s o r p tion o f con tamina ting pro teins Lyophilized, pooled Sephadex G-100 fractions containing LIF were dissolved in 2 ml 0.1 M phosphate buffer, pH 7.2, 0.5 M NaC1, applied to the immunoglobulin-Sepharose column and eluted with the same buffer at a flow rate of 0.5 ml/min. An identical column was run in parallel with pooled fractions from Sephadex G-100 chromatographed control supernatant. The initial 2.5 ml eluate was discarded and the following 8 ml were collected, desalted on Sephadex G-50 and lyophilized. Lyophilized material from both chromatographed and non-chromatographed supernatants was reconstituted in medium TC-199 (Difco Labor., Michigan, U.S.A.) containing 10% horse serum to give the concentrations of the original supernatants outlined in table 1. These preparations were tested for LIF activity on the same indicator cell population. Protein measurements The method of L o w r y et al. (1951) was employed using human serum albumin as standard. RESULTS Immunoelectrophoresis Fig. l a shows the immunoprecipitation pattern obtained when concentrated lymphokine containing crude supernatant was run in CIE against antisup. Essentially the same pattern was seen when control supernatants were tested. The two sharp and normally stained precipitates migrate together with and anodic of albumin in the first dimension and show the characteristic features of partial immunochemical identity. The unending anodic arc of the blurred precipitate (b) is also indicative of partial identity with c. Three precipitates with a and ~ mobility are distinguishable b u t stain poorly and diffusely. Precipitates with double peaks, unending and blurred precipitates and changes in migration velocity are all features of proteolytic degradation (Bjerrum and B~bg-Hansen, 1975). Since the use of aprotinin, a protease inhibitor, has been shown to inhibit an artefact in CIE caused by proteolytic activity in antibody preparations (Bjerrum et al., 1975) several experiments were carried o u t with and w i t h o u t aprotinin in the antibody solution. This, however, did n o t change the precipitation pattern. When performing CIE with gels containing the non-ionic detergent Berol, the findings were also unchanged. The visible effects of proteolytic degradation did n o t increase as a function of time, since identical precipitation patterns were obtained when testing supernatants after 6 h, 24 h and 72 h at 23°C following the initial culture period.
326
When concentrated supernatants from both stimulated and unstimulated l y m p h o c y t e s were run against an antibody preparation against human serum proteins eleven precipitates appeared (fig. l b ) . Using CIE with intermediate gels containing monospecific antibodies against serum proteins all except three precipitates were identified. It appeared that only albumin was degraded by proteolytic activity (fig.2). These degraded molecules shared at least two antigenic determinants recognized by the monospecific antibody against native albumin. This antibody preparation also contained immunoglobulins against the three antigens visible in a, b and c in fig. la. When subjected to Sephadex G-100 column chromatography all three components eluted in the same fractions corresponding to a molecular weight of 70,000 daltons. The anti-sup, preparation did not contain detectable antibodies to super-
q
.B
Fig. 1. Crossed immunoelectrophoresis of concentrated serum-free supernatants from human lymphocytes stimulated with Con A. A. Antigen: 20 pl supernatant (x 50). Antibodies: 15 pl/cm 2 of rabbit antibodies against human lymphocyte supernatant. B. Antigen: 15 pl supernatant.(× 100). Antibodies: 7.5 pl/cm 2 of pooled rabbit antibodies against human serum proteins. The following precipitates were identified: prealbumin (p), albumin (a, b and c), orosomucoid (o), alfal-antitrypsin (at), haptoglobin (h) and transferrin (t). The arrows indicate the features of partial immunochemical identity. The 5 × 5 cm plates were stained with Coomassie Brilliant Blue R.
327
Fig. 2. Crossed immunoelectrophoresis of 15 pl concentrated (X 100) serum-free supernatants from human lymphocytes stimulated with Con A. A. Blank intermediate gel. B. Intermediate gel containing absorbed monospecific antibodies against human albumin (5 pl/cm2). The reference gels contained antibodies against human serum proteins (7.5 pl/cm2). Precipitates of albumin and degraded products of albumin are marked a, b and c. The 5 X 7 cm plates were stained with Coomassie Brilliant Blue R.
n a t a n t p r o t e i n s which were n o t p r e s e n t in c o m m e r c i a l anti-whole serum protein (fig. 3). H o w e v e r , separate e x p e r i m e n t s s h o w e d t h a t the a n t i b o d y titer o f anti-sup, against c o m p o n e n t c (fig. l a ) s e e m e d to be m u c h higher t h a n t h a t o f anti-whole serum p r o t e i n . T h e p r o t e i n c o n c e n t r a t i o n o f u n c o n c e n t r a t e d s u p e r n a t a n t s f r o m b o t h stim u l a t e d and u n s t i m u l a t e d l y m p h o c y t e s was 38 p g / m l (standard deviation: 9 ttg/ml, 6 e x p e r i m e n t s ) and 33 p g / m l (standard deviation: 7 pg/ml, 6 experim e n t s ) respectively. T h u s , the c o n c e n t r a t i o n o f i d e n t i f i e d p r o t e i n s o t h e r t h a n native and degraded albumin m u s t be v e r y low. H o w e v e r , a n t i b o d i e s against some o f these m o l e c u l e s were p r o d u c e d b y the i m m u n i z e d animals (fig. 4).
Immunosorption of protein contaminants F o u r d i f f e r e n t e x p e r i m e n t s were carried o u t using p o o l e d Con A stimulated s u p e r n a t a n t s c o r r e s p o n d i n g t o original v o l u m e s o f 145 ml to 200 ml. E q u a l a m o u n t s o f c o n t r o l s u p e r n a t a n t s were r u n in parallel.
328
Fig. 3. Crossed i m m u n o e l e c t r o p h o r e s i s of 20 ~l c o n c e n t r a t e d (× 50) serum-free supernatants f r o m h u m a n l y m p h o c y t e s stimulated with Con A. The intermediate gel contained antibodies against h u m a n serum proteins (10 pl/cm2), and the reference gel contained antibodies against h u m a n l y m p h o c y t e supernatant (15 pl/cm2). No precipitates are detectable in the reference gel. The 5 × 7 cm plate was stained with Coomassie Brilliant Blue R.
A
B
Fig. 4. Crossed i m m u n o e l e c t r o p h o r e s i s of h u m a n serum. A. Blank intermediate gel. B. I n t e r m e d i a t e gel containing antibodies against h u m a n l y m p h o c y t e supernatant (50 pl/cm2). The reference gels contained antibodies against h u m a n serum proteins (17 /al/ cm2). It is shown, that the intermediate gel in B contain antibodies against the following serum proteins (arrows): albumin (a), o r o s o m u c o i d (o), a l f a r a n t i t r y p s i n (at), haptoglobin (h) and transferrin (t). The 10 × 10 cm plates were stained with Coomassie Brilliant Blue R.
329
4:,
Fig. 5. Crossed immunoelectrophoresis of pooled, LIF-rich, Sephadex G-100 fractions before (A) and after (B) affinity chromatography for immunological removal of contaminating proteins. A. Antigen: 152pl pooled Sephadex G-100 fractions (× 70 original supernatant). Antibodies: 7.5 pl/cm of pooled rabbit antibodies against human serum proteins. B. Antigen: 15 pl affinity chromatographed solution (X 70 original supernatant). Antibodies: As in 1A. The 5 X 5 cm plates were stained with Coomassie Brilliant Blue R.
The recovery of LIF activity could be estimated by testing stepwise concentrated chromatographed and non-chromatographed material on the same indicator cell population. As shown in table 1 a high yield of LIF was obtained after immunosorption of contaminating proteins. LIF activity could n o t be demonstrated in control supernatants when compared with cells migrating in medium alone. The protein contents of both active and control supernatants are also shown in table 1. Following immunosorption of contaminants the total amount of protein in the active supernatants was reduced by a factor of 23 to 53. Approximately the same relative reduction in protein contents of active and control supernatants was found. Crossed immunoelectrophoresis of Sephadex G-100 chromatographed, LIF-rich material run against a balanced antibody preparation to human serum proteins revealed two major reactions and one very faint precipitate (fig. 5a). These precipitates were identified as albumin and degraded products of albumin. Passage through the immunoglobulin-Sepharose column removed all three precipitating proteins (fig. 5b). DISCUSSION
The partial purification of human LIF seems to present even more difficulties than encountered during the attempts to purify guinea pig MIF (Remold et al., 1970; Rocklin, 1975). This is mainly because LIF, unlike MIF, cannot be separated from albumin because of their similarity in molecular weight and electrophoretic mobility. Proteolytic enzymes are frequently active during the preparation of tissue proteins (Barrett and Dingle, 1971). Supernatants of antigen-stimulated lym-
330
phocytes also contain proteases, and an esterase associated with MIF and LIF activity has been proposed (Haveman et al., 1972; Remold, 1974). Since some lymphokines are unstable in solution even when frozen, degradation by contaminating proteases is possible, and measures to inhibit these enzymes might be beneficial in preserving lymphokines. However, the expression of human LIF activity seems to be inhibited b y some animal and plant antiproteases and phenylmethyl sulfonyl fluoride (Bendtzen, 1976). The use of antiproteases in l y m p h o c y t e cultures is therefore n o t feasible for obtaining more stable lymphokine preparations. The experiments reported here were initiated in order to provide more information on the nature of contaminants with special emphasis towards proteins with similar electrophoretic mobility to that of LIF. With this information an affinity chromatography technique was developed for rapid removal of all detectable protein contaminants. Since even very small amounts of supernatant proteins gave rise to antib o d y production undetected proteins must contribute little to the total amount of contaminants. N o t all proteins, however, may induced the formation of precipitating antibodies and be demonstrable by immunoelectrophoresis, but the use of affinity column chromatography might nevertheless remove all contaminants to which an antibody response occured. Since antibodies to LIF were apparently n o t elicited by the immunization schedule used, such a technique could prove useful in attempts to purify LIF. Immunoglobulins to whole serum proteins and antibodies against crude LIFrich supernatants were therefore covalently b o u n d to a beaded agarose matrix. The latter immunoglobulins were employed because they contained higher titers against albumin c o m p o n e n t c (fig. 1). As shown, the technique provides an estimated 40-fold purification of LIF, b u t even more important is the very high yield of active material making this procedure well suited as an initial step in LIF purification. The c~lumn capacity for removing contaminants is sufficiently high to enable very small columns to be used. This presumably contributes to the high recovery of mediator activity by reducing the amount of unspecifically adsorbed material. The procedure is rapid and the risk of protein denaturation seems negligible. Furthermore, the experiments can be carried out at low cost since the columns can be regenerated and used several times. Thus, the principle of direct immunological removal of contaminating protein seems promising for obtaining high purity lymphokine preparations. It must however be realized that the technique presented here has limited efficiency, and should be combined with other methods in attempts to isolate specific lymphokines. ACKNOWLEDGEMENTS The study was supported by grants from the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Greenland and the Faroe
331
Islands, the Danish Medical Research Council and the Danish Foundation for the Advancement of Medical Science. I thank the staff of The Protein Laboratory, University of Copenhagen for valuable aid and Mrs. Lidy Broersma for expert technical assistance. REFERENCES Axelsen, N.H., 1973, Scand. J. Immunol. 2, Suppl. 1, 71. Barrett, A.J. and J. Dingle, 1971, Tissue Proteinases (North-Holland, Amsterdam-London). Bendtzen, K., 1975, Acta Allergol. 30, 327. Bendtzen, K., 1976, Acta Pathol. Microbiol. Scand. Sect. C. (in press). Bendtzen, K., V. Andersen and G. Bendixen, 1975, Acta Allergol. 30, 133. Bjerrum, O.J. and T.C. BOg-Hansen, 1975, Scand. J. Immunol. 4, Suppl. 2, 89. Bjerrum, O.J., J. Ramlau, I. Clemmesen, A. Ingild and T.C. B~bg-Hansen, 1975, Scand. J. Immunol. 4, Suppl. 2, 81. Clausen, J.E., 1972, J. Immunol. 108,453. Dumonde, D.C., D.A. Page, M. Matthew and R.A. Wolstencroft, 1972, Clin. Exp. Immunol. 10, 25. Granger, G.A., E.C. Laserna, W.P. Kolb and F. Chapman, 1973, Proc. Natl. Acad. Sci. 70, 27. Harboe, N. and A. Ingild, 1973, Scand. J. Immunol. 2, Suppl. 1, 161. Havemann, K., M. Horvat, C.-P. Sodomann, K. Havemann and S. Biirger, 1972, Eur. J. Immunol. 2, 97. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Remold, H.G., 1974, J. Immunol. 112, 1571. Remold, H.G., A.B. Katz, E. Haber and J.R. David, 1970, Cell. Immunol. 1,133. Rocklin, R.E., 1975, J. Immunol. 114, 1161. Weeke, B., 1973a, Scand. J. Immunol. 2, Suppl. 1, 15. Weeke, B., 1973b, Scand. J. Immunol. 2, Suppl. 1, 47.
